Characterization of the glycosaminoglycan-binding region of lactoferrin.
Characterization of the glycosaminoglycan-binding region of lactoferrin.
复制标题
乳铁蛋白糖胺聚糖结合区域的表征。
DOI：
10.1006/abbi.1995.1139
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Church,FC
中科院分区：
文献类型：
--
作者：
Wu,HF;Monroe,DM;Church,FC
乳铁蛋白是中性粒细胞次级颗粒的主要成分，在某些炎性疾病中其血药浓度升高。虽然乳铁蛋白作为铁结合蛋白的生化特性已经得到很好的描述，但其在炎症中的生理作用仍然不确定。我们研究了乳铁蛋白调节糖胺聚糖加速的凝血酶-丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）反应的能力。乳铁蛋白有效降低了三种生理性糖胺聚糖（包括肝素、硫酸乙酰肝素和硫酸皮肤素）引起的凝血酶-丝氨酸蛋白酶抑制剂（抗凝血酶和肝素辅因子II）反应的速率。酶动力学分析表明，乳铁蛋白没有改变肝素催化的凝血酶-抗凝血酶反应的表观肝素-凝血酶或肝素-抗凝血酶解离常数值。然而，饱和时的最大反应速度相对于任一蛋白质显着降低乳铁蛋白。采用金黄色葡萄球菌V8蛋白酶对乳铁蛋白进行有限的蛋白水解，分析乳铁蛋白的糖胺聚糖结合区。根据对肝素-琼脂糖凝胶的亲和性，纯化了相对分子质量分别为1.8kDa和1.11kDa的乳铁蛋白片段。氨基酸序列分析表明，这两个肽来自N-末端。尽管与完整的乳铁蛋白相比能力稍差，但乳铁蛋白肽有效地中和肝素、硫酸乙酰肝素和硫酸皮肤素催化的丝氨酸蛋白酶抑制剂-凝血酶抑制反应。此外，乳铁蛋白N-末端肽对肝素-琼脂糖凝胶的结合亲和力与完整乳铁蛋白的结合亲和力大致相同。检查的N-末端氨基酸序列和晶体结构的乳铁蛋白进一步支持的结论，乳铁蛋白是一种新的糖胺聚糖结合蛋白和推定的糖胺聚糖结合位点定位于N-末端。
Lactoferrin is a prominent component of neutrophil secondary granules and its blood concentration is increased in certain inflammatory diseases. Although the biochemical characterization of lactoferrin as an iron-binding protein has been well described, its physiological role in inflammation remains undefined. We examined the ability of lactoferrin to regulate glycosaminoglycan-accelerated thrombin-serine protease inhibitor (serpin) reactions. Lactoferrin effectively reduced the rate of thrombin-serpin (antithrombin and heparin cofactor II) reactions by three physiological glycosaminoglycans including heparin, heparan sulfate, and dermatan sulfate. An enzyme kinetics analysis showed that lactoferrin did not alter the apparent heparin-thrombin or the heparin-antithrombin dissociation constant values for the heparin-catalyzed thrombin-antithrombin reaction. However, the maximum reaction velocity at saturation with respect to either protein was markedly decreased by lactoferrin. The glycosaminoglycan-binding region of lactoferrin was analyzed following limited proteolysis using Staphylococcus aureus V8protease. Two lactoferrin fragments with Mr′s of ∼8 and ∼11 kDa were purified based on their affinity to heparin-Sepharose. Amino acid sequence analysis demonstrated that both peptides were from the N-terminus. Although slightly less capable compared to intact lactoferrin, the lactoferrin peptides effectively neutralized heparin, heparan sulfate, and dermatan sulfate-catalyzed serpin-thrombin inhibition reactions. In addition, lactoferrin N-terminal peptides have approximately the same binding affinity to heparin-Sepharose as that of intact lactoferrin. Inspection of both the N-terminal amino acid sequence and the crystal structure of lactoferrin further supports the conclusion that lactoferrin is a novel glycosaminoglycan binding protein and that the putative glycosaminoglycan-binding site is localized to the N-terminus.